WO2016110214A1 - 一种抗蓝耳病克隆猪的制备方法 - Google Patents

一种抗蓝耳病克隆猪的制备方法 Download PDF

Info

Publication number
WO2016110214A1
WO2016110214A1 PCT/CN2015/099835 CN2015099835W WO2016110214A1 WO 2016110214 A1 WO2016110214 A1 WO 2016110214A1 CN 2015099835 W CN2015099835 W CN 2015099835W WO 2016110214 A1 WO2016110214 A1 WO 2016110214A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
porcine
exon
seq
vector
Prior art date
Application number
PCT/CN2015/099835
Other languages
English (en)
French (fr)
Inventor
李宁
胡晓湘
陈婧瑶
Original Assignee
中国农业大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国农业大学 filed Critical 中国农业大学
Priority to US15/542,330 priority Critical patent/US20190284580A1/en
Publication of WO2016110214A1 publication Critical patent/WO2016110214A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0273—Cloned animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
    • A61D19/00—Instruments or methods for reproduction or fertilisation
    • A61D19/04—Instruments or methods for reproduction or fertilisation for embryo transplantation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • C12N15/877—Techniques for producing new mammalian cloned embryos
    • C12N15/8778—Swine embryos
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0337—Animal models for infectious diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites

Definitions

  • the invention belongs to the field of animal genetic engineering and genetic genetic modification, and in particular relates to a CD163 gene modification method for anti-blue ear cloning pigs and a preparation method for anti-blue ear cloning pigs by using the CRISPR/Cas9 system.
  • Porcine reproductive and respiratory syndrome also known as blue ear disease
  • PRRSV porcine reproductive and respiratory syndrome virus
  • PRRSV porcine reproductive and respiratory syndrome virus
  • PRRSV porcine reproductive and respiratory syndrome virus
  • the disease first appeared in the United States in 1987, followed by the outbreak in Europe in 1989, and gradually spread to other parts of the world.
  • the frequent outbreak of PRRS in the world has caused huge economic losses.
  • the “pigless high fever” caused by PRRSV mutant strains in China and Vietnam has caused the pig industry in both countries to suffer.
  • PRRSV mainly infects porcine alveolar macrophages (PAM) in the body and can also infect peripheral blood mononuclear cells and sperm cells.
  • PAM porcine alveolar macrophages
  • MA104 African green monkey kidney cell line MA104 and its derived cell line MARC-145 are currently infected.
  • the study found that there are three receptors for PRRSV, heparin sulphate (HS), sialoadhesin (Sn) and CD163 (cluster of differentiation 163) on PAM.
  • HS heparin sulphate
  • Sn sialoadhesin
  • CD163 cluster of differentiation 163
  • the virus After the virus is endocytosed, it quickly enters the early inclusion bodies, and the genome of the virus is released into the cytoplasm. This process relies on the acidification of inclusion bodies and CD163, and cathepsin E and a trypsin-like serine protease that has not yet been identified also play a role in this process.
  • the CD163 molecule consists of nine repetitive scavenger receptor-rich cysteine domains (SRCR domain), two interdomain sequences (PSTI, PSTII), one transmembrane sequence, and one cytoplasmic tail.
  • SRCR domain repetitive scavenger receptor-rich cysteine domains
  • PSTI two interdomain sequences
  • PSTI two interdomain sequences
  • CD163 cytoplasmic tail.
  • Van Gorp's team prepared cells expressing mutant CD163.
  • SRCR5 of CD163 is necessary for infection, and at the N-terminal four SRCR domains and cytoplasm The tail is unnecessary.
  • Phani B. Das et al. found that the envelope glycoprotein GP4 of PRRSV mediates the formation of the envelope polyprotein complex and works with GP2a.
  • the ligand of CD163 molecule plays an important role in the process of viral endocytosis.
  • the CRISPR (clustered regular interspaced short palindromic repeats)/Cas (CRISPR-associated) system is a prokaryotic immune system specific to exogenous genetic material. It cleaves and degrades exogenous DNA through sequence-specific RNA mediated, including Phage and foreign plasmids.
  • the CRISPR/Cas system can be used as a site-specific gene editing system. Its biggest features are simple operation, low cost and high efficiency. In 2013, scientists first reported the successful application of the CRISPR/Cas system on cells, and subsequently rapidly applied in zebrafish, fruit flies, mice, rats, and pigs.
  • the CRISPR/Cas system produces a double strand break (DSB) at the target site, and the cells can be repaired by non-homologous end joining (NHEJ), resulting in a frameshift mutation and loss of function. .
  • NHEJ non-homologous end joining
  • the system can work with homologous recombination vectors and oligonucleotides to efficiently and accurately modify target genes.
  • Scott JG et al. used CRISPR/Cas-mediated homologous recombination to achieve gene replacement on zebrafish.
  • Hui Yang et al. used the same strategy to obtain a mouse with a reporter gene.
  • the CRISPR/Cas system has rapidly become a leader in gene editing tools because of its great advantages, and has been widely used in gene function research, disease models, gene therapy and other fields.
  • CRISPR/Cas9-mediated homologous recombination has enabled precise editing of genes in zebrafish, protozoa, mice, and rats, but has not been reported in large livestock animals.
  • the object of the present invention is to provide a method for preparing a cloned pig against blue ear disease by using a CRISPR-Cas9 system to modulate homologous recombination to modify the porcine CD163 gene to obtain a cloned pig resistant to blue ear disease.
  • the present invention first provides the use of exon 7 of the porcine CD163 gene for the preparation of cloned pigs against blue ear disease.
  • the nucleotide sequence of exon 7 of the porcine CD163 gene is shown in SEQ ID NO.
  • the present invention provides a porcine CD163 gene homologous recombination modification vector, wherein the 7th exon of the porcine CD163 gene is replaced with the 10th exon of the human CD163-L1 gene, and the 7th exon of the porcine CD163 gene
  • the sub-nucleotide sequence is shown in SEQ ID NO. 1, and the 10th exon nucleotide sequence of the human CD163-L1 gene is shown, for example, in SEQ ID NO.
  • the porcine CD163 gene homologous recombination modification vector provided by the invention is prepared by the following method:
  • the homologous right arm nucleotide sequence of the exon 7 of the porcine CD163 gene of the above step (1) is as shown in SEQ ID NO. 3; the exon 7 of the porcine CD163 gene of step (2) is homologous to the left arm.
  • the nucleotide sequence is shown in SEQ ID NO.
  • the primer sequence for amplifying the homologous right arm of exon 7 of the porcine CD163 gene is set forth in SEQ ID NO.
  • Primer sequences for amplifying the homologous left arm of exon 7 of the porcine CD163 gene are set forth in SEQ ID NO.
  • the primer sequence for amplifying the 10th exon of the human CD163-L1 gene is shown in SEQ ID NO.
  • the present invention also provides an sgRNA which specifically targets the exon 7 of the porcine CD163 gene, the sequence of which is (as shown in SEQ ID NO. 5).
  • the oligonucleotide sequence complementary thereto is (as shown in SEQ ID NO. 6).
  • the present invention also provides another sgRNA which specifically targets the exon 7 of the porcine CD163 gene, and the sequence thereof is (as shown in SEQ ID NO. 7).
  • the oligonucleotide sequence complementary thereto is (as shown in SEQ ID NO. 8).
  • the present invention also provides a CRISPR/Cas9 targeting vector containing the DNA sequence of the above sgRNA.
  • the CRISPR/Cas9 targeting vector of the present invention is prepared by the following method, and the oligonucleotides shown in SEQ ID NO. 5 and 6 are placed at 94 ° C, 5 min, 35 ° C, 10 min, and then immediately iced.
  • the oligonucleotide was annealed; the px330 backbone vector was digested with restriction endonuclease BbsI overnight, and after recovery, the CRISPR/Cas9 targeting vector of the present invention was obtained by ligation with the annealed oligonucleotide.
  • the invention provides a method for preparing a cloned pig against blue ear disease, which comprises transferring a CRISPR/Cas9 targeting vector and a porcine CD163 gene homologous recombination modified vector into a pig fibroblast to obtain a positive cell clone;
  • the cells are nuclear donor donor cells, and the oocytes are nuclear transfer recipient cells.
  • the cloned embryos are obtained by somatic cell nuclear transfer technology; the cloned embryos are transferred into the pig uterus for pregnancy to obtain CD163 gene-modified anti-blue ear cloned pigs.
  • the CRISPR/Cas9 targeting vector and the porcine CD163 gene homologous recombination modified vector were co-transformed into porcine fibroblasts: CRISPR/Cas9 targeting vector and porcine CD163 gene homologous recombination modified carrier total mass 4-6 ⁇ g, according to the amount of substance 1 A ratio of 1:1 was mixed and transferred to about 1 x 10 6 pig fibroblasts by electroporation or lipofection.
  • the present invention utilizes CRISPR/Cas9 technology to mediate homologous recombination for the first time in large animals, making endogenous sources
  • the CD163 gene was precisely edited (see Figure 1).
  • This method is low cost, greatly shortens the time to obtain homozygous pigs, and ensures that CD163 expression is not affected by gene editing, and uses homologous recombination for large animals using CRISPR/Cas9 technology. It laid the foundation for gene function research and disease model establishment.
  • FIG. 1 is a schematic diagram showing the replacement of exon 7 of the endogenous CD163 gene of pig with the exon 10 of human CD163L1 gene in Example 1 of the present invention.
  • Fig. 2 is a flow chart showing the construction of a porcine CD163 gene-modified vector in Example 1 of the present invention.
  • Fig. 3 is a diagram showing the porcine CD163 gene-modified vector obtained in Example 1 of the present invention.
  • Figure 4 is a diagram showing the cleavage of the genome of the px330 plasmid on porcine embryo fibroblasts in the porcine embryo fibroblasts in Example 2 of the present invention, and 501 and 502 respectively represent the two px330 plasmids obtained by the present invention.
  • Fig. 5 is a diagram showing the identification of cell monoclonals by the PCR method in Example 3 of the present invention, wherein Figs. 5a, 5b, and 5c respectively show steps 1, 2, and 3 in the process. 1, 2, 3, 4, and 5 in the figure indicate the obtained positive cell monoclonals numbered 1, 2, 3, 4, and 5, respectively.
  • Fig. 6 is a PCR method for identifying newborn pigs in Example 4 of the present invention, wherein Figs. 6a, 6b, and 6c respectively show steps 1, 2, and 3 in the process.
  • Figure 7 is a graph showing the identification of homozygous clones of newborn cloned pigs by the sequencing method in Example 4 of the present invention.
  • Figure 8 is a diagram showing the transcription of the CD163 gene in the tissues of newborn cloned pigs after qRT-PCR identification in Example 4 of the present invention.
  • Figure 9 is a diagram showing the expression of the CD163 gene in the tissues of newborn cloned pigs by Western blot in Example 4 of the present invention.
  • Figure 10 is a graph showing the infection of alveolar macrophages in cloned pigs and wild-type pigs after different doses of challenge in Example 5, wherein Figure 10a is a picture of cytopathic effect after challenge of WUH3 strain, and Figure 10b is a strain of JXwn06.
  • the picture of cytopathic lesions after challenge, Figure 10c and Figure 10d are the relative expression of viral RNA and the viral titer in the supernatant after challenge with JXwn06 strain, respectively.
  • Figure 10e and Figure 10f show the relative viral RNA after challenge of WUH3 strain, respectively. The amount of expression and the viral titer in the supernatant.
  • Figure 11 is a diagram showing the detection of infection of alveolar macrophages in cloned pigs and wild-type pigs at different time points in a challenge dose according to Example 5 of the present invention, wherein Figures 11a, 11b and 11c are respectively JXwn06 The relative expression of viral RNA, viral titer and viral protein expression in the supernatant after challenge. Figure 11d shows the relative expression of viral RNA after challenge with the WUH3 strain.
  • the px330 vector and LoxPneoLoxP2PGK vector were purchased from Addgene; T4 DNA ligase and restriction endonuclease were purchased from Dalian TaKaRa; primer synthesis and sequencing were completed by Shanghai Shengong and Shenzhen Huada; KOD DNA polymerase was purchased from Shanghai Dongyang Spinning Co., Ltd.
  • the human CD163-L1 exon 10 (the nucleotide sequence is shown as SEQ ID NO. 2) and the homologous right arm were fused.
  • the exon 10 of human CD163L1 was amplified, and the primers used for amplification were with The PCR product was purified and verified by sequencing.
  • a porcine cell genomic DNA as a template, a 999 bp homologous right arm was amplified (the nucleotide sequence is shown in SEQ ID NO. 3), and the primer was with Underlined for the SalI restriction site, sequencing confirmed correctly.
  • the 10th exon of human CD163L1 gene was fused with the homologous right arm and named as fusion fragment 1. The sequencing was verified.
  • Step 2 Using the porcine cell genomic DNA as a template, a 6392 bp homologous left arm (nucleotide sequence as shown in SEQ ID NO. 4) was amplified, and the primer was with Underlined for the SacII restriction site, sequencing confirmed correctly. The fusion fragment 1 was fused with the homologous left arm and designated as fusion fragment 2, and the sequencing was verified correctly.
  • Step 3 The vector LoxPneoLoxP2PGK was digested with SalI and SacII restriction enzymes at 37 ° C to recover larger fragments. The recovered fragment was ligated with the restriction fragment 2 (SalI and SacII) and ligated with T4 DNA ligase at 16 ° C to obtain a final porcine CD163 gene-modified vector, as shown in FIG.
  • Two targeting vectors were constructed, named px330-501 and px330-502.
  • the two pairs of oligonucleotides in Table 1 were used respectively.
  • the construction process was as follows: 94 ° C, 5 min, then 35 ° C, 10 min, then immediately placed On ice, the oligonucleotide is annealed.
  • the px330 backbone vector was digested with restriction endonuclease BbsI overnight, and after recovery, it was ligated with the annealed oligonucleotide at 16 ° C for 3 h. Conversion and plating are carried out by a conventional conversion method. After the single colony grows, several expanded cultures are picked and sequenced. The sequencing verification was correct, indicating that the present invention successfully constructed two CRISPR-Cas9 targeting vectors.
  • the specific steps are as follows: a. Initial culture, pick a positive single colony with a pipette tip, and add a sterilization tube containing 5 ml of LB medium (peptone 10 g, yeast extract 5 g, NaCl 10 g dissolved in 1 L of distilled water), 37 °C, 220 rpm, culture for 8h to 12h; b. Expand the culture, transfer the overnight culture solution to a volumetric 1:500 ratio to a sterile flask containing 100ml of LB medium, 37 ° C, 220 rpm, culture 12h to 16h;
  • the px330 plasmid was extracted according to the method provided on the EndoFree Plasmid Maxi Kit, and the resulting plasmid was used for cell transfection.
  • Cell transfection was performed by Lonza Nucleofector. The specific procedure is as follows: a. Mix one hole of porcine fibroblasts (about 1 ⁇ 10 6 ), 4 ⁇ g of px330 plasmid and 100 ⁇ l of Nucleofector reagent in a 6-well cell culture plate that has been digested and collected, and put it into a shock cup with T- 016 procedure for electric shock transfection; b. After the shock is completed, slowly add 500 ⁇ L of 37°C pre-warmed fibroblast culture medium (10% FBS+DMEM) along the inner wall of the electric shock cup, and inoculate the cells in one well of the 6-well cell culture plate. c; cultured in fibroblast culture medium (10% FBS + DMEM) without screening drug in a 37.5 ° C, 5% CO 2 incubator.
  • fibroblast culture medium (10% FBS + DMEM
  • the cell genome was extracted according to the method provided on the Dneasy Blood & Tissue Kit. Using the extracted genome and wild-type porcine fibroblast genome as a template, PCR was performed with KOD DNA polymerase to amplify a 785 bp fragment. with The amplification conditions were 94 ° C, 2 min; 94 ° C, 30 sec; 60 ° C, 30 sec; 68 ° C, 60 sec; 68 ° C, 7 min; 35 cycles, 1.0% agarose electrophoresis, and then the PCR product was recovered and the concentration was measured. 400 ng of PCR recovered product was annealed and cooled from the 95 ° C program to 4 ° C.
  • the annealed product was digested with T7E1 enzyme at 37 ° C for 1 h.
  • the system was: annealed product 10 ⁇ l, NEB buffer 2 2 ⁇ l, T7E1 0.5 ⁇ l, and ddH 2 O was made up to 20 ⁇ l. After digestion, the results were observed by PAGE gel electrophoresis. Both px330 plasmids could play a role in cutting the genome on porcine fibroblasts, as shown in Figure 4.
  • the porcine fibroblasts (about 1 ⁇ 10 6 ) in one well of a 6-well cell culture plate were digested and collected, and the targeting vector px330-501 constructed in Example 2 and the porcine CD163 gene constructed in Example 1 were homologously recombined.
  • the carrier was mixed at a ratio of 1:1 in the amount of the substance, and the total mass was 4 ⁇ g.
  • the transfection was carried out in the same manner as in the step 5 of Example 2, and then placed in a CO 2 incubator and cultured at 37.5 °C. After 48 hours, the cell confluence reached 80-90%. At this time, the cells of one well were equally divided into 8 10 cm culture dishes.
  • the cells were adherent, and the medium was changed to a fibroblast culture medium (10% FBS + DMEM) containing G418 (600 ⁇ g/mL), and the solution was changed every 3 to 4 days, and the medium was still containing G418 (600 ⁇ g/mL). Fibroblast culture medium. After 6 to 9 days of cell culture, cell clone point formation was observed. Find the resistant cell clones under the microscope, mark them with a Marker pen, pour off the medium, wash once with PBS solution, cover the resistant cell clones with the cell clone loop, and add 10-30 ⁇ L of pre-heated 0.25 at 37 °C.
  • a fibroblast culture medium (10% FBS + DMEM) containing G418 (600 ⁇ g/mL)
  • the solution was changed every 3 to 4 days, and the medium was still containing G418 (600 ⁇ g/mL).
  • Fibroblast culture medium After 6 to 9 days of cell culture, cell clone point formation was observed
  • % trypsin digest digest the cells at 37.5 ° C for about 2 min, add the cell culture medium to terminate the digestion reaction, and inoculate the digested cells into a 48-well cell culture plate.
  • the cells are digested and inoculated into a 12-well cell culture plate to continue the culture.
  • the undigested cells in the original 48-well cell culture plate are further cultured for extracting the genomic DNA of the cells, and the cells continue to expand.
  • the cells were cultured in a 6-well cell culture plate, and the resistant cells were cryopreserved according to the method of cryopreservation of porcine embryonic fibroblasts.
  • PCR was carried out by KOD DNA polymerase to amplify a 703 bp fragment, and the primer was with
  • the PCR reaction using wild-type porcine fibroblast genomic DNA as a template was a negative control.
  • Amplification conditions 94 ° C, 2 min; 94 ° C, 30 sec; 60 ° C, 30 sec; 68 ° C, 60 sec; 68 ° C, 7 min; 35 cycles. After the amplification was completed, the results were observed by 1.0% agarose electrophoresis.
  • the PCR product was recovered by gel cutting and the concentration was measured.
  • the purified PCR product was digested with BbsI restriction enzyme, digested at 37 ° C for 4 h, and observed by 1.5% agarose electrophoresis, as shown in Figure 5a. Since the human CD163L1 exon 10 contains a BbsI cleavage site and the porcine CD163 exon 7 does not contain it, BbsI can cleave the 703 bp PCR product into two segments if recombination occurs.
  • the second step of the cell monoclonal identified as positive in the first step is identified.
  • PCR was carried out using Q5 DNA polymerase to amplify a 1317 bp fragment, and the primer was with
  • the PCR reaction using wild-type porcine fibroblast genomic DNA as a template was a negative control.
  • Amplification conditions 98 ° C, 30 sec; 98 ° C, 10 sec; 64 ° C, 30 sec; 72 ° C, 45 sec; 72 ° C, 2 min; 35 cycles. After the amplification was completed, the results were observed by 1.0% agarose electrophoresis, and the results are shown in Fig. 5b.
  • the upstream primer 39-F used in this step is located on the 10th exon of human CD163L1, and the downstream primer 40-R is located downstream of the homologous right arm. Therefore, if homologous recombination occurs, the 1317 bp fragment can be amplified, if not. Recombination cannot be amplified.
  • step 3 the cell monoclonal identified as positive in step 2 was subjected to step 3 identification.
  • PCR was performed using LongAmp Taq DNA polymerase to amplify a 6897 bp fragment. with The PCR reaction using wild type cell genomic DNA as a template was a negative control.
  • Amplification conditions 94 ° C, 30 sec; 94 ° C, 30 sec; 60 ° C, 30 sec; 65 ° C, 16 min; 65 ° C, 10 min; 35 cycles. After the amplification was completed, the results were observed by 0.8% agarose electrophoresis. Eight of the 54 cell clones were positive for monoclonal, and the results are shown in Figure 5c.
  • the upstream primer 43-F used in this step is located upstream of the homologous left arm, and the downstream primer 44-R is located on the 10th exon of human CD163L1. Therefore, if homologous recombination occurs, a fragment of 6897 bp can be amplified, if not. Recombination did not amplify a 6897 bp fragment.
  • the first step of the identification indicates that the exon 10 of human CD163L1 is inserted into the genome of porcine fibroblasts, and the results of the second and third steps indicate that the insertion position is correct, and the positive step can be determined by the three-step identification step.
  • the positive cells successfully obtained by the homologous recombination obtained in Example 3 are nuclear transfer donor cells, and the in vitro mature primordial sow oocytes are used as nuclear transfer recipient cells, and the nuclear transfer donor cells are transferred into the enucleated cells.
  • the oocyte is electrofused and activated to construct a cloned embryo.
  • the cloned embryo with good morphology is selected and transferred to the uterus of the natural estrus to perform pregnancy.
  • the surgical embryo transfer procedure is Shutai routine anesthesia.
  • supine Baoding On the surgical frame, supine Baoding, a midline of the abdomen to make a surgical incision about 8cm long, expose the ovary, fallopian tube and uterus, use the embryo transfer tube to enter the ear canal along the fallopian tube about 5cm, transplant the embryo (more than 300) to the oviduct ampulla - isthmus Meeting point.
  • B-mode ultrasound was used to detect pregnancy or not 30 days after embryo transfer.
  • the cloned pig born in the full-term pregnancy in step 1 was subjected to PCR detection, and the detection method was consistent with the detection method of the cell monoclonal in Example 3.
  • the results are shown in Fig. 6a, Fig. 6b and Fig. 6c respectively.
  • the PCR products of primers CD7tF and CD7tR were completely cut by BbsI. Further sequencing revealed that the peak map was single, as shown in Fig. 7, indicating that the newborn pig was homozygous.
  • the transcription of CD163 in various tissues of cloned pigs was detected by qRT-PCR. Take the cloned pig and wild type pig liver, spleen, lung, small intestine and other four tissues, each taking 30-50mg, add 1ml Trizol, and homogenize with steel beads for 10min. After the homogenization was completed, 0.2 ml of chloroform was added, and the mixture was homogenized for 15 sec, and allowed to stand at room temperature for 2 min. Centrifuge at 12000 rpm for 15 min at 4 °C. At this time, the sample was divided into three layers: red organic phase, intermediate layer and upper colorless aqueous phase. RNA was mainly in the aqueous phase.
  • the aqueous phase (about 600 ⁇ l) was transferred to a new centrifuge tube, and the like.
  • the volume of isopropanol was mixed upside down and allowed to stand at room temperature for 10 min. Centrifuge at 12000 rpm for 15 min at 4 ° C and discard the supernatant. The precipitate was washed by adding 1 ml of 75% ethanol and washed a total of two times. Centrifuge at 12000 rpm for 3 min at 4 °C, carefully discard the supernatant, taking care not to aspirate the RNA pellet. After standing at room temperature for 2 min, 30 ⁇ l of RNase-free water was added to fully dissolve the RNA precipitate.
  • RNA 2 ⁇ g was taken and reverse transcribed with reverse transcriptase to obtain cDNA.
  • the system for real-time fluorescence quantification was as follows: SYBR Green 7.5 ⁇ l, upstream and downstream primers 0.2 ⁇ l, template cDNA 1 ⁇ l, and ddH 2 O supplemented to 15 ⁇ l. Primer is with The fluorescence quantitation used was a Roche LightCycler 480 fluorescence quantitative PCR machine. The results of qRT-PCR showed that the transcriptional trend of CD163 in the tissues of the newly cloned pigs in which the CD163 gene was modified was consistent with the wild type, as shown in FIG.
  • cloned pig liver, spleen and lung tissues were taken, and 100 mg of liver, spleen and lung tissues of wild type pigs were used as negative controls.
  • the tissue was cut into fine pieces and 1 ml of lysate was added (the lysate was added to PMSF several minutes before use to give a final concentration of PMSF of 1 mM). Homogenize with a glass homogenizer until fully cleaved. After sufficient lysis, centrifugation was carried out at 12,000 g for 3 minutes, and the supernatant was taken to obtain total protein of each tissue.
  • the alveolar macrophages of cloned pigs and wild-type pigs were respectively resuscitated into one well of a 6-well plate. After 24 hours of culture, the medium was removed, washed once with PBS, 200 ⁇ l of lysate was added to each well, and the number was beaten and centrifuged at 12000 g for 3 minutes. The supernatant was taken to obtain the total protein of alveolar macrophages. The concentration was measured using a BCA protein concentration assay kit. 20 ⁇ g of total protein was taken and electrophoresed on a 6% SDS-PAGE gel, 60 V, 1 h; 90 V, 2 h.
  • the Bio-Rad wet transfer membrane was used for 350 mA and the membrane was transferred for 80 min. After the transfection was completed, it was blocked with 5% skim milk powder overnight, then rabbit anti-porcine primary antibody (1:300 dilution) was incubated for 2 h, TBST was washed for 3 ⁇ 10 min, and then HRP-labeled goat anti-rabbit secondary antibody (1:10000 dilution) Incubation for 1 h, TBST wash membrane for 3 ⁇ 10 min, and finally BCL color development. The results are shown in Fig. 9.
  • the expression level of CD163 in the tissues of the cloned pigs with anti-blue ear disease obtained in the present invention is not different from that in the wild type pigs, indicating that the expression of the anti-Blue ear disease cloned pig CD163 of the present invention is not affected by the CD163 gene.
  • the expression of the exon is replaced by the 10th exon of the human CD163-L1 gene.
  • Anatomically cloned pigs and SPF (Specific Pathogen Free) wild-type control pigs take lungs in a clean environment (clamp the pig's trachea with a hemostat before cutting the trachea), and perform lung lavage in a clean bench; HBSS buffer (with penicillin and streptomycin) was slowly instilled into the lungs (about 500-800 ml each time), and each time the lungs were filled with lungs, all the lavage fluid was collected and collected by centrifugation at 400g.
  • HBSS buffer with penicillin and streptomycin
  • the cells were collected and washed with RPMI 1640 medium (plus double antibody); the washing was repeated twice; the cells were collected, the cells were counted, the cells were resuspended in the cell cryopreservation solution, and the cells were frozen (about 10 7 per tube). ⁇ 10 8 cells).
  • the cells were first resuscitated at the time of use and then cultured with RPMI 1640 medium containing 10% FBS. When the virus infects the cells, a certain amount of the virus is first adsorbed to the cells for 1 hour (a small amount of the culture solution), and the culture medium is further added to continue the culture.
  • the cloned porcine PAM cells and wild-type porcine PAM cells were challenged with different doses of JXwn06 strain and WUH3 strain.
  • the cytopathic lesions were observed 48 hours after challenge, and the supernatant was assayed for virus by TCID 50 method.
  • the titer and the cell-extracted RNA were detected by Real-Time PCR.
  • the anti-Blue ear disease cloned porcine PAM cells obtained by the present invention have the ability to resist PRRSV at different challenge doses, and reach a level of complete protection. Subsequently, the cytopathic effect at different time points after challenge was observed under a dose of the challenge.
  • the virus titer was also determined by the TCID 50 method, and the virus was collected by Real-Time PCR.
  • the anti-Blue ear disease cloned porcine PAM cells obtained by the present invention have the ability to resist PRRSV at different time points after the same dose of challenge, and reach a level of complete protection.
  • the invention provides a method for preparing a cloned pig against blue ear disease, which utilizes a CRISPR-Cas9 system to modulate homologous recombination to modify the porcine CD163 gene to obtain a cloned pig against blue ear disease.
  • the method has low cost, greatly shortens the time for obtaining homozygous pigs, and ensures that the expression of CD163 is not affected by gene editing, which lays a foundation for large animal to use cDNA/Cas9 technology to mediate homologous recombination for gene function research and disease model establishment. .

Abstract

提供一种抗蓝耳病克隆猪的制备方法，采用CRISPR/Cas9打靶载体与CD163基因同源重组修饰载体共转入猪成纤维细胞中，获得阳性细胞克隆，阳性克隆中的猪内源CD163基因的第7个外显子被替换为人CD163-L1基因的第10外显子，使其不具有介导PRRSV入侵的能力；以阳性细胞为核移植供体细胞，卵母细胞为核移植受体细胞，通过体细胞核移植技术获得克隆胚胎；将克隆胚胎移入猪子宫内妊娠获得克隆猪。

Description

一种抗蓝耳病克隆猪的制备方法 技术领域
本发明属于动物基因工程和基因遗传修饰领域，具体地说，涉及一种利用CRISPR/Cas9系统进行的抗蓝耳病克隆猪的CD163基因修饰方法以及抗蓝耳病克隆猪的制备方法。
背景技术
猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome，PRRS)又称蓝耳病，是由猪繁殖与呼吸综合征病毒(PRRSV)引起的以妊娠母猪繁殖障碍及各年龄阶段猪呼吸道症状为主要特征的传染病，并引起严重的免疫抑制。该病最早于1987年在美国出现，紧接着于1989年在欧洲爆发，并从此逐渐向世界其它地区扩散。PRRS在世界范围内的频繁爆发造成了巨大的经济损失，如PRRSV突变株在中国和越南引起的“猪无名高热病”导致两国的养猪业遭受重创。
PRRSV在体内主要感染猪肺泡巨噬细胞(porcine alveolar macrophages，PAM)，也能感染外周血单核细胞和精子细胞。在体外，目前能感染非洲绿猴肾细胞系MA104及其衍生细胞系MARC-145。研究发现，在PAM上存在PRRSV的3个受体，硫酸乙酰肝素(heparin sulphate，HS)、唾液酸黏附素(sialoadhesin，Sn)和CD163(cluster of differentiation 163)分子。PRRSV最先与PAM表面的HS接触，随后转换成与Sn发生更加稳定的互作。Sn与病毒粘附后，病毒-受体复合体在网格蛋白的介导下发生内吞。病毒被内吞后很快进入到早期的包涵体中，病毒的基因组被释放到细胞质中。这个过程依赖于包涵体的酸化作用和CD163，组织蛋白酶E和一种还没有鉴定清楚的胰蛋白酶样丝氨酸蛋白酶也在这个过程中起作用。
CD163分子由9个重复的清道夫受体富含半胱氨酸结构域(SRCR结构域)、2个域间序列(PSTⅠ、PSTⅡ)、1个跨膜序列和1个细胞质尾巴组成。CD163被鉴定为PRRSV感染PAM的必需受体之后，研究者开始探索CD163的哪些结构域是其作为受体所必需的。2010年，Van Gorp研究小组制备了表达突变型CD163的细胞，通过检测这些细胞与PRRSV的粘附能力发现，CD163的SRCR5对于感染是必要的，同时，N端的4个SRCR结构域和细胞质中的尾巴是不必要的。同年，Phani B.Das等的研究发现，PRRSV的囊膜糖蛋白GP4介导囊膜多聚蛋白复合体的形成，并且和GP2a一起作为 CD163分子的配体，在病毒内吞过程中起重要作用。
CRISPR(clustered regularly interspaced short palindromic repeats)/Cas(CRISPR-associated)系统是一种原核生物特有的针对外源性遗传物质的免疫系统，通过序列特异的RNA介导，切割降解外源性DNA，包括噬菌体和外源质粒。CRISPR/Cas系统可以作为一种具有位点特异性的基因编辑系统，其最大的特点是操作简单、成本低、作用高效。2013年，科学家首次报道CRISPR/Cas系统在细胞上应用成功，随后，在斑马鱼、果蝇、小鼠、大鼠、猪中迅速得到应用。CRISPR/Cas系统在靶位点产生双链DNA断裂(double strand break,DSB),细胞可通过非同源末端连接(non-homologous end joining,NHEJ)进行修复，导致基因发生移码突变，丧失功能。除此之外，该系统还能与同源重组载体、寡聚核苷酸共同作用，使靶基因发生高效的精确修饰。2014年，Scott JG等利用CRISPR/Cas介导的同源重组在斑马鱼上实现了基因的替换。Hui Yang等利用相同的策略一步获得了带有报告基因的小鼠。CRISPR/Cas系统凭借其巨大优势迅速成为基因编辑工具中的佼佼者，在基因功能研究、疾病模型、基因治疗等领域得到广泛的应用。
CRISPR/Cas9介导同源重组已在斑马鱼、原虫、小鼠、大鼠上实现基因的精确编辑，但在家畜大动物上尚未见报道。
发明内容
本发明的目的是提供一种抗蓝耳病克隆猪的制备方法，是利用CRISPR-Cas9系统介导同源重组修饰猪CD163基因以获得抗蓝耳病克隆猪。
本发明首先提供了猪CD163基因第7外显子在制备抗蓝耳病克隆猪中的用途。所述猪CD163基因第7外显子的核苷酸序列如SEQ ID NO.1所示。
本发明提供了一种猪CD163基因同源重组修饰载体，该载体上猪CD163基因的第7外显子被替换为人CD163-L1基因的第10外显子，所述猪CD163基因的第7外显子核苷酸序列如SEQ ID NO.1所示，所述人CD163-L1基因的第10外显子核苷酸序列例如SEQ ID NO.2所示。
本发明提供的猪CD163基因同源重组修饰载体，通过如下方法制备得到：
(1)将人CD163-L1第10外显子和猪CD163基因第7外显子同源右臂融合，得到融合片段1；
(2)将融合片段1与猪CD163基因第7外显子同源左臂融合，得到融合片段2；
(3)用SalⅠ和SacⅡ限制性内切酶分别对融合片段2和载体LoxPneoLoxP2PGK进行双酶切，然后连接得到猪CD163基因同源重组修饰载体。
其中，上述步骤(1)的猪CD163基因第7外显子同源右臂核苷酸序列如SEQ ID NO.3所示；步骤(2)的猪CD163基因第7外显子同源左臂核苷酸序列如SEQ ID NO.4所示。
在本发明的实施例中，用于扩增猪CD163基因第7外显子同源右臂的引物序列如SEQ ID NO.9、10所示。用于扩增猪CD163基因第7外显子同源左臂的引物序列如SEQ ID NO.11、12所示。用于扩增人CD163-L1基因的第10外显子的引物序列如SEQ ID NO.13、14所示
本发明还提供了特异性靶向猪CD163基因第7外显子的sgRNA，其序列为
Figure PCTCN2015099835-appb-000001
(如SEQ ID NO.5所示)。与其互补配对的寡核苷酸序列为
Figure PCTCN2015099835-appb-000002
(如SEQ ID NO.6所示)。
本发明还提供了另一个特异性靶向猪CD163基因第7外显子的sgRNA，其序列为
Figure PCTCN2015099835-appb-000003
(如SEQ ID NO.7所示)。与其互补配对的寡核苷酸序列为
Figure PCTCN2015099835-appb-000004
(如SEQ ID NO.8所示)。
本发明还提供了含有上述sgRNA的DNA序列的CRISPR/Cas9打靶载体。
本发明所述的CRISPR/Cas9打靶载体，其通过以下方法制备得到，将SEQ ID NO.5、6所示的寡聚核苷酸在94℃，5min，再35℃，10min，然后立即放冰上对寡聚核苷酸进行退火；px330骨架载体用限制性内切酶BbsⅠ进行酶切过夜，回收后，与退火的寡聚核苷酸连接即得本发明的CRISPR/Cas9打靶载体。
本发明提供了一种抗蓝耳病克隆猪的制备方法，是将权利要求CRISPR/Cas9打靶载体与猪CD163基因同源重组修饰载体共转入猪成纤维细胞中，获得阳性细胞克隆；以阳性细胞为核移植供体细胞，卵母细胞为核移植受体细胞，通过体细胞核移植技术获得克隆胚胎；将克隆胚胎移入猪子宫内妊娠获得CD163基因修饰后的抗蓝耳病克隆猪。
CRISPR/Cas9打靶载体与猪CD163基因同源重组修饰载体共转入猪成纤维细胞的方法为：CRISPR/Cas9打靶载体和猪CD163基因同源重组修饰载体总质量4-6μg，按物质的量1:1的比例混合，用电击转染或脂质体转染的方法转入约1×106个猪成纤维细胞。
本发明首次在大动物上利用CRISPR/Cas9技术介导同源重组，使内源 CD163基因得以精确编辑(见图1)，该方法成本低，大幅缩短获得纯合子猪的时间，并保证CD163的表达不受基因编辑的影响，为大动物利用CRISPR/Cas9技术介导同源重组进行基因功能研究及疾病模型建立奠定了基础。
附图说明
图1是本发明实施例1中将猪内源CD163基因第7外显子替换为人CD163L1基因第10外显子的示意图。
图2是本发明实施例1中猪CD163基因修饰载体的构建流程图。
图3是本发明实施例1中获得的猪CD163基因修饰载体图。
图4是本发明实施例2中T7E1酶切法鉴定在猪胚胎成纤维细胞上px330质粒对基因组的切割情况，501、502分别表示本发明获得的两个px330质粒。
图5是本发明实施例3中PCR法鉴定细胞单克隆，其中图5a、图5b、图5c分别表示该过程中的第1、2、3步。图中1、2、3、4、5分别表示获得的编号为1号、2号、3号、4号、5号的阳性细胞单克隆。
图6是本发明实施例4中PCR法鉴定新生猪，其中图6a、图6b、图6c分别表示该过程中的第1、2、3步。
图7是本发明实施例4中测序法鉴定新生克隆猪纯合子情况的峰图。
图8是本发明实施例4中qRT-PCR鉴定改造后的CD163基因在新生克隆猪各组织中的转录情况。
图9是本发明实施例4中Western blot鉴定改造后的CD163基因在新生克隆猪各组织中的表达情况。
图10是本发明实施例5中不同剂量攻毒后克隆猪和野生型猪肺泡巨噬细胞感染情况的检测，其中图10a是WUH3毒株攻毒后的细胞病变图片，图10b是JXwn06毒株攻毒后的细胞病变图片，图10c和图10d分别是JXwn06毒株攻毒后病毒RNA相对表达量和上清中病毒滴度，图10e和图10f分别是WUH3毒株攻毒后病毒RNA相对表达量和上清中病毒滴度。
图11是本发明实施例5中一个攻毒剂量下的不同时间点克隆猪和野生型猪肺泡巨噬细胞感染情况的检测，其中图11a、图11b、图11c分别是JXwn06 毒株攻毒后病毒RNA相对表达量、上清中病毒滴度和病毒蛋白表达情况。图11d是WUH3毒株攻毒后病毒RNA相对表达量。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
px330载体、LoxPneoLoxP2PGK载体购于Addgene公司；T4DNA连接酶、限制性内切酶购于大连TaKaRa公司；引物合成及序列测定由上海生工和深圳华大完成；KOD DNA聚合酶购于上海东洋纺公司；LongAmp Taq DNA聚合酶、Q5DNA聚合酶、T7E1酶购于NEB公司；Trizol Reagent购于康为世纪公司；反转录酶购于Promega公司；SYBR Green购于Roche公司；质粒去内毒素大提试剂盒、基因组提取试剂盒购于QIAGEN公司；酶切、连接、回收、转化、PCR扩增等常规实验操作步骤详见《分子克隆(第三版)》。
实施例1猪CD163基因修饰载体的构建
该载体构建过程共分为三步，过程见图2：
第1步，将人CD163-L1第10外显子(核苷酸序列如SEQ ID NO.2所示)和同源右臂融合。以人细胞的基因组DNA为模板，扩增出人CD163L1第10外显子，扩增所采用的引物为
Figure PCTCN2015099835-appb-000005
和
Figure PCTCN2015099835-appb-000006
Figure PCTCN2015099835-appb-000007
纯化PCR产物并测序验证正确。以猪细胞基因组DNA为模板，扩增出999bp的同源右臂(核苷酸序列如SEQ ID NO.3所示)，引物为
Figure PCTCN2015099835-appb-000008
Figure PCTCN2015099835-appb-000009
和
Figure PCTCN2015099835-appb-000010
Figure PCTCN2015099835-appb-000011
下划线为SalⅠ酶切位点，测序验证正确。将人CD163L1基因第10外显子与同源右臂进行融合，命名为融合片段1，测序验证正确。
第2步：以猪细胞基因组DNA为模板，扩增出6392bp的同源左臂(核苷酸序列如SEQ ID NO.4所示)，引物为
Figure PCTCN2015099835-appb-000012
和
Figure PCTCN2015099835-appb-000013
Figure PCTCN2015099835-appb-000014
下划线为SacⅡ 酶切位点，测序验证正确。将融合片段1与同源左臂进行融合，命名为融合片段2，测序验证正确。
第3步：将载体LoxPneoLoxP2PGK用SalⅠ和SacⅡ限制性内切酶于37℃进行消化，回收较大的片段。回收得到的片段与酶切(SalⅠ和SacⅡ)后的融合片段2用T4DNA连接酶于16℃进行连接，得到最终的猪CD163基因修饰载体，见图3。
实施例2 CRISPR-Cas9打靶载体的构建
1、利用张锋实验室网站(http://crispr.genome-engineering.org/)对猪CD163基因第7外显子的打靶位点进行预测。根据自我评估和预测结果中的评分，从候选的靶位点中选择两个，命名为501、502，其sgRNA序列分别为
Figure PCTCN2015099835-appb-000015
和
Figure PCTCN2015099835-appb-000016
根据sgRNA序列合成互补配对的寡聚核苷酸，如表1所示，其中小写字母为酶切位点。
表1寡聚核苷酸序列
Figure PCTCN2015099835-appb-000017
2、共构建2个打靶载体，命名为px330-501、px330-502，分别使用表1的两对寡聚核苷酸，构建过程如下：94℃，5min，再35℃，10min，然后立即放冰上，对寡聚核苷酸进行退火。px330骨架载体用限制性内切酶BbsⅠ进行酶切过夜，回收后，与退火的寡聚核苷酸16℃连接3h。通过常规转化法进行转化、涂板。待单菌落长成后，挑取数个扩大培养并测序。测序验证正确，说明本发明成功构建两个CRISPR-Cas9打靶载体。
3、阳性单菌落扩大培养
具体步骤为：a.初始培养，用枪头挑取阳性单菌落，加入盛有5ml LB培养基(胰蛋白胨10g、酵母提取物5g、NaCl 10g溶于1L蒸馏水中)的灭菌管中，37℃，220rpm，培养8h至12h；b.扩大培养，将过夜培养液按体积1:500的比例转移到盛有100ml LB培养基的灭菌三角瓶中，37℃，220rpm，培养12h至16h；
4、px330质粒去内毒素大提
按照质粒去内毒素大提试剂盒(EndoFree Plasmid Maxi Kit)上提供的方法，提取px330质粒，所提的质粒用于细胞的转染。
5、细胞转染
细胞转染采用Lonza Nucleofector进行电转。具体流程如下：a.将消化并收集的6孔细胞培养板中一个孔的猪成纤维细胞(约1×106个)、4μg px330质粒和100μl Nucleofector试剂混匀，装入电击杯用T-016程序进行电击转染；b.电击结束后，沿电击杯内壁缓慢加入37℃预热的成纤维细胞培养基(10％FBS+DMEM)500μL，将细胞接种于6孔细胞培养板的一个孔内；c.用无筛选药物的成纤维细胞培养基(10％FBS+DMEM)于37.5℃，5％CO2培养箱培养。
6、打靶效率的检测
按照基因组提取试剂盒(Dneasy Blood&Tissue Kit)上提供的方法，提取细胞基因组。以提取的基因组和野生型猪成纤维细胞基因组为模板，用KOD DNA聚合酶进行PCR，扩增出785bp的片段，引物为
Figure PCTCN2015099835-appb-000018
Figure PCTCN2015099835-appb-000019
和
Figure PCTCN2015099835-appb-000020
Figure PCTCN2015099835-appb-000021
扩增条件为94℃，2min；94℃，30sec；60℃，30sec；68℃，60sec；68℃，7min；35个循环，1.0％琼脂糖电泳观察结果，然后回收PCR产物，测浓度。取400ng PCR回收产物进行退火，从95℃程序降温至4℃。退火后的产物用T7E1酶进行酶切，37℃1h，体系为：退火产物10μl，NEB buffer2 2μl，T7E1 0.5μl，ddH2O补足至20μl。酶切完成后用PAGE胶电泳观察结果，两个px330质粒均能在猪成纤维细胞上发挥切割基因组的作用，如图4所示。
实施例3阳性细胞单克隆的筛选及鉴定
1、阳性单克隆细胞的筛选
消化并收集6孔细胞培养板中一个孔的猪成纤维细胞(约1×106个)，将实施例2构建的打靶载体px330-501和实施例1构建得到的猪CD163基因同源重组修饰载体，按物质的量1:1的比例混合，取总质量4μg，按实施例2中的步骤5的方法进行转染后，放至CO2培养箱中，37.5℃培养。48h后细胞汇合度达到80-90％，此时将1个孔的细胞平均分到8个10cm培养皿中。24h后细胞贴壁，将培养基更换为含G418(600μg/mL)的成纤维细胞培养基 (10％FBS+DMEM)，每3～4d换一次液，培养基仍然为含G418(600μg/mL)的成纤维细胞培养基。细胞培养6～9d后，可以观察到细胞克隆点形成。在显微镜下找到抗性细胞克隆点，用Marker笔做标记，倒掉培养基，PBS溶液清洗一次，用细胞克隆环将抗性细胞克隆点罩住，加入10～30μL在37℃预热的0.25％胰蛋白酶消化液，37.5℃消化细胞2min左右，加入细胞培养基终止消化反应，将消化下来的细胞接种到48孔细胞培养板中培养。待细胞汇合度达到90％时，消化细胞，接种到12孔细胞培养板中继续培养，原来48孔细胞培养板中的未被消化下来的细胞也继续培养以供提取细胞基因组DNA，细胞继续扩大培养至6孔细胞培养板，按照猪胚胎成纤维细胞冻存方法进行抗性细胞冻存。
2、阳性细胞单克隆的鉴定
对所挑取的54个细胞单克隆进行鉴定：
第1步，以提取的细胞单克隆基因组DNA为模板，用KOD DNA聚合酶进行PCR，扩增出703bp的片段，引物为
Figure PCTCN2015099835-appb-000022
Figure PCTCN2015099835-appb-000023
和
Figure PCTCN2015099835-appb-000024
Figure PCTCN2015099835-appb-000025
以野生型猪成纤维细胞基因组DNA为模板的PCR反应为阴性对照。扩增条件：94℃，2min；94℃，30sec；60℃，30sec；68℃，60sec；68℃，7min；35个循环。扩增完成后，1.0％琼脂糖电泳观察结果。切胶回收PCR产物，测浓度。纯化后的PCR产物用BbsⅠ限制性内切酶进行酶切消化，37℃消化4h，1.5％琼脂糖电泳观察结果，见图5a。由于人CD163L1第10外显子上含有BbsⅠ的酶切位点，而猪CD163第7外显子上不含有，因此若发生重组，BbsⅠ能将703bp的PCR产物切割成两段。
第2步，对第一步鉴定为阳性的细胞单克隆进行第二步鉴定。用Q5DNA聚合酶进行PCR，扩增出1317bp的片段，引物为
Figure PCTCN2015099835-appb-000026
Figure PCTCN2015099835-appb-000027
和
Figure PCTCN2015099835-appb-000028
Figure PCTCN2015099835-appb-000029
以野生型猪成纤维细胞基因组DNA为模板的PCR反应为阴性对照。扩增条件：98℃，30sec；98℃，10sec；64℃，30sec；72℃，45sec；72℃，2min；35个循环。扩增完成后，1.0％琼脂糖电泳观察结果，结果见图5b。该步鉴定所用的上游引物39-F位于人CD163L1第10外显子上，下游引物40-R位于同源右臂下游，因此若发生同源重组则能扩增出1317bp的片段，若没发生重组则不能扩增出。
第3步，对第2步鉴定为阳性的细胞单克隆进行第3步鉴定。用LongAmp Taq DNA聚合酶进行PCR，扩增出6897bp的片段，引物为
Figure PCTCN2015099835-appb-000030
Figure PCTCN2015099835-appb-000031
和
Figure PCTCN2015099835-appb-000032
Figure PCTCN2015099835-appb-000033
以野生型细胞基因组DNA为模板的PCR反应为阴性对照。扩增条件：94℃，30sec；94℃，30sec；60℃，30sec；65℃，16min；65℃，10min；35个循环。扩增完成后，0.8％琼脂糖电泳观察结果。54个细胞单克隆中有8个为阳性单克隆，结果见图5c，结果图中显示状态适宜做核移植的5个克隆，其中5号克隆为阳性。该步鉴定所用的上游引物43-F位于同源左臂上游，下游引物44-R位于人CD163L1第10外显子上，因此若发生同源重组则能扩增出6897bp的片段，若没发生重组则不能扩增出6897bp的片段。
本实施例中，第1步鉴定结果说明人CD163L1第10外显子插入到了猪成纤维细胞的基因组中，第2、3步鉴定结果均说明插入位置正确，通过这3步鉴定步骤能够确定阳性细胞单克隆。
实施例4 CD163基因修饰的抗蓝耳病克隆猪的制备与鉴定
1、CD163基因修饰抗蓝耳病克隆猪的制备
以实施例3获得的成功发生同源重组的阳性细胞为核移植供体细胞，以体外成熟的初情期前母猪卵母细胞为核移植受体细胞，将核移植供体细胞移入去核的卵母细胞，经电融合与激活，构建成克隆胚胎，挑选形态优良的克隆胚胎用手术法移入自然发情的经产母猪子宫内进行妊娠,手术法胚胎移植步骤为舒泰常规麻醉,在手术架上仰卧保定,腹中线做一个长约8cm的手术切口,曝露卵巢、输卵管及子宫,用胚胎移植管沿输卵管伞部进入约5cm,将胚胎(300枚以上)移植到输卵管壶腹部－峡部结合处。胚胎移植后30天B型超声波检测妊娠与否。
2、新生猪的PCR检测
对步骤1中妊娠足月出生的克隆猪进行PCR检测，检测方法与实施例3中细胞单克隆的检测方法一致。结果分别如图6a、图6b、图6c所示，引物CD7tF、CD7tR的PCR产物被BbsⅠ完全切开，进一步的测序发现峰图单一，如图7所示，说明新生猪为纯合子。
3、新生猪的qRT-PCR检测
用qRT-PCR检测克隆猪各种组织中CD163的转录情况。取克隆猪和野 生型猪肝、脾、肺、小肠等四种组织，每种取30-50mg，加1ml Trizol，用钢珠匀浆10min。匀浆结束后，加入0.2ml氯仿，再匀浆15sec，室温放置2min。4℃12000rpm离心15min，此时样品分三层：红色有机相、中间层和上层无色水相，RNA主要在水相，把水相(约600μl)转移到一个新的离心管中，加入等体积的异丙醇，颠倒混匀，室温放置10min。4℃12000rpm离心15min，弃上清。加入1ml 75％乙醇洗涤沉淀，共洗两次。4℃12000rpm离心3min，小心吸弃上清，注意不要吸弃RNA沉淀。室温放置2min后加入30μl RNase-free的水，充分溶解RNA沉淀。取2μg RNA，用反转录酶进行反转录，得到cDNA。实时荧光定量的体系如下：SYBR Green 7.5μl，上下游引物分别0.2μl，模板cDNA 1μl，加ddH2O补足至15μl。引物为
Figure PCTCN2015099835-appb-000034
Figure PCTCN2015099835-appb-000035
和
Figure PCTCN2015099835-appb-000036
Figure PCTCN2015099835-appb-000037
所用的荧光定量仪为Roche LightCycler 480荧光定量PCR仪。qRT-PCR的结果显示CD163基因被修饰的新生克隆猪各组织中CD163的转录趋势与野生型一致，如图8所示。
4、克隆猪的Western blot检测
取克隆猪肝、脾、肺组织各100mg，野生型猪肝、脾、肺组织各100mg作为阴性对照。把组织剪切成细小的碎片，加入1ml裂解液(裂解液在使用前数分钟加入PMSF，使PMSF的最终浓度为1mM)。用玻璃匀浆器匀浆，直至充分裂解。充分裂解后，12000g离心3分钟，取上清获得各组织总蛋白。分别复苏克隆猪和野生型猪的肺泡巨噬细胞至6孔板的一个孔中，培养24h后去除培养基，用PBS洗一遍，每孔加入200μl裂解液，吹打数下，12000g离心3分钟，取上清获得肺泡巨噬细胞总蛋白。用BCA蛋白浓度测定试剂盒测浓度。各取20μg总蛋白，用6％的SDS-PAGE凝胶，60V,1h；90V，2h进行电泳。电泳完毕后利用Bio-Rad湿转转膜仪350mA，转膜80min。转膜完成后，用5％脱脂奶粉封闭过夜，然后兔抗猪一抗(1:300稀释)进行孵育2h，TBST洗膜3×10min，然后HRP标记的羊抗兔二抗(1：10000稀释)孵育1h，TBST洗膜3×10min,最后进行BCL显色。结果如图9所示，本发明获得的抗蓝耳病克隆猪各组织CD163的表达量与野生型猪没有差异，说明本发明的抗蓝耳病克隆猪CD163的表达不受CD163基因第7外显子被替换为人CD163-L1基因的第10外显子的影响。
5、克隆猪PAM细胞攻毒试验
解剖克隆猪和SPF(Specific Pathogen Free)野生型对照猪，在尽量干净 的环境中取肺(在切断气管前用止血钳夹住猪的气管)，在超净工作台中进行肺部灌洗；用HBSS缓冲液(加青霉素和链霉素)分批次缓慢灌入肺中(大约每次500～800ml)，每次对灌满的肺进行肺部按摩，收集所有的灌洗液，400g离心收集细胞；收集的细胞用RPMI 1640培养液(加双抗)进行洗涤；重复洗涤2次；收集细胞，进行细胞计数，用细胞冻存液重悬细胞，进行分装冻存(每管约107～108个细胞)。细胞在使用时先进行复苏，然后用含有10％FBS的RPMI 1640培养液进行培养。病毒在感染细胞时，一定量的病毒先与细胞吸附1小时(少量培养液)，再补加培养液继续培养。首先采用不同的攻毒剂量的JXwn06毒株和WUH3毒株对克隆猪PAM细胞和野生型猪PAM细胞进行攻毒，在攻毒后48小时观察细胞病变，收上清液用TCID50法测定病毒滴度，并收细胞提取RNA采用Real-Time PCR的方法对病毒含量进行检测。结果如图10所示，本发明获得的抗蓝耳病克隆猪PAM细胞在不同攻毒剂量下均具有抵抗PRRSV的能力，并且达到完全保护的水平。随后在一个攻毒剂量下观察攻毒后不同时间点的细胞病变，同样也收上清液用TCID50法测定病毒滴度，收细胞提取RNA采用Real-Time PCR的方法对病毒含量进行检测。结果如图11所示，本发明获得的抗蓝耳病克隆猪PAM细胞在同一剂量攻毒后的不同时间点均具有抵抗PRRSV的能力，并且达到完全保护的水平。
虽然，上文中已经用一般性说明、具体实施方式及试验，对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供一种抗蓝耳病克隆猪的制备方法，利用CRISPR-Cas9系统介导同源重组修饰猪CD163基因以获得抗蓝耳病克隆猪。该方法成本低，大幅缩短获得纯合子猪的时间，并保证CD163的表达不受基因编辑的影响，为大动物利用CRISPR/Cas9技术介导同源重组进行基因功能研究及疾病模型建立奠定了基础。
Figure PCTCN2015099835-appb-000038
Figure PCTCN2015099835-appb-000039
Figure PCTCN2015099835-appb-000040
Figure PCTCN2015099835-appb-000041
Figure PCTCN2015099835-appb-000042

Claims (10)

  1. 猪CD163基因第7外显子在制备抗蓝耳病克隆猪中的用途，所述猪CD163基因第7外显子的核苷酸序列如SEQ ID NO.1所示。
  2. 猪CD163基因同源重组修饰载体，其特征在于，猪CD163基因的第7外显子被替换为人CD163-L1基因的第10外显子，所述猪CD163基因的第7外显子核苷酸序列如SEQ ID NO.1所示，所述人CD163-L1基因的第10外显子核苷酸序列例如SEQ ID NO.2所示。
  3. 如权利要求2所述的猪CD163基因同源重组修饰载体，其特征在于，通过如下方法制备得到：
    (1)将人CD163-L1第10外显子和猪CD163基因第7外显子同源右臂融合，得到融合片段1；
    (2)将融合片段1与猪CD163基因第7外显子同源左臂融合，得到融合片段2；
    (3)用Sal Ⅰ和Sac Ⅱ限制性内切酶分别对融合片段2和载体LoxPneoLoxP2PGK进行双酶切，然后连接得到猪CD163基因同源重组修饰载体。
  4. 如权利要求3所述的猪CD163基因同源重组修饰载体，其特征在于，步骤(1)的猪CD163基因第7外显子同源右臂核苷酸序列如SEQ ID NO.3所示；步骤(2)的猪CD163基因第7外显子同源左臂核苷酸序列如SEQ ID NO.4所示。
  5. 如权利要求3所述的猪CD163基因同源重组修饰载体，其特征在于，用于扩增猪CD163基因第7外显子同源右臂的引物序列如SEQ ID NO.9、10所示；用于扩增猪CD163基因第7外显子同源左臂的引物序列如SEQ ID NO.11、12所示；用于扩增人CD163-L1基因的第10外显子的引物序列如SEQ ID NO.13、14所示。
  6. 特异性靶向猪CD163基因第7外显子的sgRNA，其特征在于，其DNA序列如SEQ ID NO.5所示或如SEQ ID NO.7所示。
  7. 含有权利要求6所述sgRNA的DNA序列的CRISPR/Cas9打靶载体。
  8. 如权利要求7所述的CRISPR/Cas9打靶载体，其通过以下方法制备得到，将SEQ ID NO.5、6所示的寡聚核苷酸或SEQ ID NO.7、8所示的寡聚核苷酸在94℃，5min，再35℃，10min，然后立即放冰上对寡聚核苷酸进行退火；px330骨架载体用限制性内切酶BbsⅠ进行酶切过夜，回收后，与退火的寡聚核苷酸连接。
  9. 一种抗蓝耳病克隆猪的制备方法，其特征在于，将权利要求7-8任一所述的CRISPR/Cas9打靶载体与权利要求2-5任一所述的猪CD163基因同源重组修饰载体共转入猪成纤维细胞中，获得阳性细胞克隆；以阳性细胞为核移植供体细胞，卵母细胞为核移植受体细胞，通过体细胞核移植技术获得克隆胚胎；将克隆胚胎移入猪子宫内妊娠获得CD163基因修饰后的抗蓝耳病克隆猪。
  10. 如权利要求9所述的制备方法，其特征在于，CRISPR/Cas9打靶载体与猪CD163基因同源重组修饰载体共转入猪成纤维细胞的方法为：CRISPR/Cas9打靶载体和猪CD163基因同源重组修饰载体总质量4-6μg，按物质的量1:1的比例混合，用电击转染或脂质体转染的方法转入约1×106个猪成纤维细胞。
PCT/CN2015/099835 2015-01-08 2015-12-30 一种抗蓝耳病克隆猪的制备方法 WO2016110214A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US15/542,330 US20190284580A1 (en) 2015-01-08 2015-12-30 Preparation method for anti-porcine reproductive and respiratory syndrome cloned pig

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510009543.4A CN104593422A (zh) 2015-01-08 2015-01-08 一种抗蓝耳病克隆猪的制备方法
CN201510009543.4 2015-01-08

Publications (1)

Publication Number Publication Date
WO2016110214A1 true WO2016110214A1 (zh) 2016-07-14

Family

ID=53119475

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/099835 WO2016110214A1 (zh) 2015-01-08 2015-12-30 一种抗蓝耳病克隆猪的制备方法

Country Status (3)

Country Link
US (1) US20190284580A1 (zh)
CN (1) CN104593422A (zh)
WO (1) WO2016110214A1 (zh)

Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9526784B2 (en) 2013-09-06 2016-12-27 President And Fellows Of Harvard College Delivery system for functional nucleases
US9840699B2 (en) 2013-12-12 2017-12-12 President And Fellows Of Harvard College Methods for nucleic acid editing
WO2018073237A1 (en) * 2016-10-17 2018-04-26 The University Court Of The University Of Edinburgh Swine comprising modified cd163 and associated methods
CN108103099A (zh) * 2017-12-18 2018-06-01 中山大学 一种抗蓝耳病Marc-145细胞系及其制备方法和应用
US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10091975B2 (en) 2015-08-06 2018-10-09 The Curators Of The University Of Missouri Pathogen-resistant animals having modified CD163 genes
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
CN108823248A (zh) * 2018-04-20 2018-11-16 中山大学 一种利用CRISPR/Cas9编辑陆川猪CD163基因的方法
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US11160260B2 (en) 2018-04-17 2021-11-02 The Curators Of The University Of Missouri Methods for protecting porcine fetuses from infection with porcine reproductive and respiratory syndrome virus (PRRSV)
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
CN115349494A (zh) * 2022-09-22 2022-11-18 中国农业大学 一种快速筛选高产蛋性能鸡的方法
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
CN116064557A (zh) * 2022-09-15 2023-05-05 华中农业大学 激活猪的ogfod2基因表达的制剂在制备猪抗伪狂犬病毒感染药物中的应用
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
LT3066201T (lt) 2013-11-07 2018-08-10 Editas Medicine, Inc. Su crispr susiję būdai ir kompozicijos su valdančiomis grnr
CN104593422A (zh) * 2015-01-08 2015-05-06 中国农业大学 一种抗蓝耳病克隆猪的制备方法
CN105624146B (zh) * 2015-05-28 2019-02-15 中国科学院微生物研究所 基于CRISPR/Cas9和酿酒酵母细胞内源的同源重组的分子克隆方法
CN105002198B (zh) * 2015-08-07 2018-07-06 北京蛋白质组研究中心 用于培育表达人血清白蛋白的转基因猪的成套产品及其应用
CN105087620B (zh) * 2015-08-31 2017-12-29 中国农业大学 一种过表达猪共刺激受体4‑1bb载体及其应用
CN105112449B (zh) * 2015-09-02 2018-02-06 中国农业大学 Cd28基因过表达载体及其应用
CN105463027A (zh) * 2015-12-17 2016-04-06 中国农业大学 一种高肌肉量及肥厚型心肌病模型克隆猪的制备方法
CN107177595A (zh) * 2017-06-07 2017-09-19 浙江大学 用于猪CD163基因编辑的靶向sgRNA、修饰载体及其制备方法和应用
CN107354170A (zh) * 2017-08-24 2017-11-17 中国农业科学院北京畜牧兽医研究所 一种基因敲除载体以及制备cd163基因敲除猪成纤维细胞的方法
CN107937345B (zh) * 2017-11-16 2019-01-29 山东蓝思种业股份有限公司 一种制备同时敲除cd163基因和cd13基因的猪成纤维细胞的方法
CN108753832A (zh) * 2018-04-20 2018-11-06 中山大学 一种利用CRISPR/Cas9编辑大白猪CD163基因的方法
CN109628494A (zh) * 2019-01-23 2019-04-16 华南农业大学 冠状病毒抗性克隆猪及其制备方法
CN110438155A (zh) * 2019-08-15 2019-11-12 中国农业科学院北京畜牧兽医研究所 修饰cd163基因第561位氨基酸的组合物、应用、细胞及基因编辑猪的制备方法
KR20230006827A (ko) * 2020-05-05 2023-01-11 제너스 피엘씨 Cd163의 표적 불활화를 통한 돼지 종의 건강 개선 방법
CN111647604A (zh) * 2020-06-29 2020-09-11 中国农业科学院北京畜牧兽医研究所 特异性识别猪COL1A1基因的gRNA及其生物材料、试剂盒和应用
CN113509552B (zh) * 2021-04-21 2022-02-18 华中农业大学 敲除或沉默猪的Kxd1基因在提高猪抗猪繁殖与呼吸综合征病毒中的应用
CN114774468B (zh) * 2022-04-20 2022-12-20 温氏食品集团股份有限公司 一种等位基因分子标记及抗蓝耳病猪群体组建方法
CN116769016B (zh) * 2023-06-14 2024-03-08 中农种源(深圳)科技有限公司 pAPN突变体和用于pAPN基因定点修饰的组合物及应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102715132A (zh) * 2012-05-04 2012-10-10 吉林大学 猪繁殖与呼吸综合症病毒受体cd163敲除猪及培育方法
CN103687482A (zh) * 2011-05-16 2014-03-26 密苏里大学管理者 抗猪生殖与呼吸综合征病毒的动物
CN104593422A (zh) * 2015-01-08 2015-05-06 中国农业大学 一种抗蓝耳病克隆猪的制备方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110016546A1 (en) * 2008-12-04 2011-01-20 Sigma-Aldrich Co. Porcine genome editing with zinc finger nucleases

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103687482A (zh) * 2011-05-16 2014-03-26 密苏里大学管理者 抗猪生殖与呼吸综合征病毒的动物
CN102715132A (zh) * 2012-05-04 2012-10-10 吉林大学 猪繁殖与呼吸综合症病毒受体cd163敲除猪及培育方法
CN104593422A (zh) * 2015-01-08 2015-05-06 中国农业大学 一种抗蓝耳病克隆猪的制备方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN, JINGYAO ET AL.: "Advances in Pathogenesis and Resistance Breeding of Porcine Reproductive and Respiratory Syndrome Virus", ACTA VETERINARIA ET ZOOTECHICA SINICA, vol. 44, no. 11, 30 November 2013 (2013-11-30), ISSN: 0366-6964 *
DATABASE Genbank 21 August 2010 (2010-08-21), LEE, Y. J. ET AL.: "Sus Scrofa CD 163 MRNA, Complete Cds", Database accession no. HM991330.1 *

Cited By (51)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US11920181B2 (en) 2013-08-09 2024-03-05 President And Fellows Of Harvard College Nuclease profiling system
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US10954548B2 (en) 2013-08-09 2021-03-23 President And Fellows Of Harvard College Nuclease profiling system
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US11299755B2 (en) 2013-09-06 2022-04-12 President And Fellows Of Harvard College Switchable CAS9 nucleases and uses thereof
US9999671B2 (en) 2013-09-06 2018-06-19 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US9526784B2 (en) 2013-09-06 2016-12-27 President And Fellows Of Harvard College Delivery system for functional nucleases
US10912833B2 (en) 2013-09-06 2021-02-09 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US9737604B2 (en) 2013-09-06 2017-08-22 President And Fellows Of Harvard College Use of cationic lipids to deliver CAS9
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US11053481B2 (en) 2013-12-12 2021-07-06 President And Fellows Of Harvard College Fusions of Cas9 domains and nucleic acid-editing domains
US9840699B2 (en) 2013-12-12 2017-12-12 President And Fellows Of Harvard College Methods for nucleic acid editing
US11124782B2 (en) 2013-12-12 2021-09-21 President And Fellows Of Harvard College Cas variants for gene editing
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US11578343B2 (en) 2014-07-30 2023-02-14 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10827730B2 (en) 2015-08-06 2020-11-10 The Curators Of The University Of Missouri Pathogen-resistant animals having modified CD163 genes
US10091975B2 (en) 2015-08-06 2018-10-09 The Curators Of The University Of Missouri Pathogen-resistant animals having modified CD163 genes
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11702651B2 (en) 2016-08-03 2023-07-18 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
WO2018073237A1 (en) * 2016-10-17 2018-04-26 The University Court Of The University Of Edinburgh Swine comprising modified cd163 and associated methods
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US11820969B2 (en) 2016-12-23 2023-11-21 President And Fellows Of Harvard College Editing of CCR2 receptor gene to protect against HIV infection
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11932884B2 (en) 2017-08-30 2024-03-19 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
CN108103099B (zh) * 2017-12-18 2022-03-04 中山大学 一种抗蓝耳病Marc-145细胞系及其制备方法和应用
CN108103099A (zh) * 2017-12-18 2018-06-01 中山大学 一种抗蓝耳病Marc-145细胞系及其制备方法和应用
US11160260B2 (en) 2018-04-17 2021-11-02 The Curators Of The University Of Missouri Methods for protecting porcine fetuses from infection with porcine reproductive and respiratory syndrome virus (PRRSV)
CN108823248A (zh) * 2018-04-20 2018-11-16 中山大学 一种利用CRISPR/Cas9编辑陆川猪CD163基因的方法
US11795452B2 (en) 2019-03-19 2023-10-24 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11643652B2 (en) 2019-03-19 2023-05-09 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
CN116064557A (zh) * 2022-09-15 2023-05-05 华中农业大学 激活猪的ogfod2基因表达的制剂在制备猪抗伪狂犬病毒感染药物中的应用
CN115349494A (zh) * 2022-09-22 2022-11-18 中国农业大学 一种快速筛选高产蛋性能鸡的方法

Also Published As

Publication number Publication date
CN104593422A (zh) 2015-05-06
US20190284580A1 (en) 2019-09-19

Similar Documents

Publication Publication Date Title
WO2016110214A1 (zh) 一种抗蓝耳病克隆猪的制备方法
US10240131B2 (en) Type II pseudorabies virus attenuated strain, its preparation method and application
CN107937345B (zh) 一种制备同时敲除cd163基因和cd13基因的猪成纤维细胞的方法
WO2019119521A1 (zh) 一种抗蓝耳病Marc-145细胞系及其制备方法和应用
US11240997B2 (en) Method for preparing porcine fibroblasts with both CD163 gene and CD13 gene being knocked-out
CN110079541B (zh) 一种构建冠状病毒感染性克隆的方法及其应用
CA3037451A1 (en) Swine comprising modified cd163 and associated methods
CN107267469B (zh) 纤毛蛋白嵌合型重组人b型腺病毒及其制备方法
CN107354170A (zh) 一种基因敲除载体以及制备cd163基因敲除猪成纤维细胞的方法
CN110951745A (zh) Cd163突变基因及其在抑制或阻断猪产生抗体的方法和应用
CN110257435A (zh) 一种prom1-ko小鼠模型的构建方法及其应用
US11535850B2 (en) Methods for improving the health of porcine species by targeted inactivation of CD163
CN109628414B (zh) 一种mRNA甲基转移酶缺陷型腮腺炎病毒及其制备方法和应用
CN113403337A (zh) 一种载体系统、制备猪成纤维细胞和基因编辑猪的方法
Kui et al. Pig macrophages with site-specific edited CD163 decrease the susceptibility to infection with porcine reproductive and respiratory syndrome virus
CN105505881B (zh) 一种猪伪狂犬病毒的抑制细胞系
CN113073114B (zh) 一种抗非洲猪瘟克隆猪的制备方法
MX2014015649A (es) Vectores de vacuna recombinante de herpesvirus bovino 1 (bohv-1) con baja virulencia.
WO2012135981A1 (zh) 利用锌指核酸酶敲除牛β-乳球蛋白基因的方法
HU227667B1 (en) Novel expression vectors and uses thereof
CN112094866B (zh) 一种利用SpRY-Cas9系统制备CD163基因编辑猪的方法
CN111705063B (zh) Asgr1突变基因及其在制备哺乳动物肝损伤敏感模型中的应用
CN117286162A (zh) 重组猪δ冠状病毒感染性克隆及其构建方法和应用
CN116855539A (zh) 一种同时敲除CD163、pAPN、MSTN基因抗病和品质改良猪培育方法
WO2020000464A1 (zh) 一种制备gl-r基因敲除小鼠的方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15876714

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15876714

Country of ref document: EP

Kind code of ref document: A1